Fungal promoters for expressing a gene in a fungal cell

ABSTRACT

The present invention relates to isolated fungal promoter DNA sequences, to DNA constructs, vectors, and fungal host cells comprising these promoters in operative association with coding sequences encoding polypeptides. The present invention also relates to methods for expressing a gene and/or producing a polypeptide using the new promoters isolated. The present invention also relates to methods for altering the transcription level and/or regulation of an endogenous gene using the new promoter of the invention.

FIELD OF THE INVENTION

The present invention relates to DNA sequences, in particular isolatedfungal promoters, and to DNA constructs, vectors, and fungal host cellscomprising these promoters in operative association with codingsequences encoding polypeptides. The present invention also relates tomethods for expressing a gene and/or producing a polypeptide.

BACKGROUND OF THE INVENTION

Production of a recombinant polypeptide in a fungal host cell is usuallyaccomplished by constructing an expression cassette in which the DNAcoding for the polypeptide is placed under the expression control of apromoter, suitable for the host cell. The expression cassette may beintroduced into the host cell, by plasmid- or vector-mediatedtransformation. Production of the polypeptide may then be achieved byculturing the transformed host cell under inducing conditions necessaryfor the proper functioning of the promoter contained in the expressioncassette.

For each fungal host cell, expression of a coding sequence which hasbeen introduced into the fungal host by transformation and production ofrecombinant polypeptides encoded by this coding sequence requires theavailability of functional promoters. Numerous promoters are alreadyknown to be functional in fungal host cells. There are examples ofcross-species use of promoters: the promoter of the Aspergillus nidulans(A. nidulans gpdA gene is known to be functional in Aspergillus niger(A. niger) (J Biotechnol. 1991 January; 17(1):19-33. Intracellular andextracellular production of proteins in Aspergillus under the control ofexpression signals of the highly expressed A. nidulans gpdA gene. Punt PJ, Zegers N D, Busscher M, Pouwels P H, van den Hondel C A.) Anotherexample is the A. niger beta-xylosidase xlnD promoter used in A. nigerand A. nidulans Transcriptional regulation of the xylanolytic enzymesystem of Aspergillus, van Peij, NNME, PhD-thesis LandbouwuniversiteitWageningen, the Netherlands, ISBN 90-5808-154-0 and the expression ofthe Escherichia coli beta-glucuronidase gene in A. niger, A. nidulansand Cladosporium fulvum as described in Curr Genet. 1989 March;15(3):177-80: Roberts I N, Oliver R P, Punt P J, van den Hondel C A.“Expression of the Escherichia con beta-glucuronidase gene in industrialand phytopathogenic filamentous fungi”.

However, there is still a need for improved promoters for controllingthe expression of introduced genes, for controlling the level ofexpression of endogenous genes, for controlling the regulation ofexpression of endogenous genes or for mediating the inactivation of anendogenous gene, or for producing polypeptides, or for combination ofthe previous applications. These improved promoters may for example bestronger than the previous known ones. They may also be inducible by aspecific convenient substrate or compound. Knowing several functionalpromoters is also an advantage when one envisages to simultaneously overexpress various genes in a single fungal host. To prevent squelching(titration of specific transcription factors), it is preferable to usemultiple distinct promoters, one specific promoter for each gene to beexpressed.

DESCRIPTION OF THE FIGURES

FIG. 1 depicts the plasmid map of pGBTOPGLA, which is an integrativeglucoamylase expression vector.

FIG. 2 depicts the plasmid map of pGBTOPGLA-2, which is an integrativeglucoamylase expression vector with a multiple cloning site.

FIG. 3 depicts the plasmid map of pGBTOPGLA-3, which is an integrativeexpression vector containing the promoter of the invention in operativeassociation with the glucoamylase coding sequence.

FIG. 4 depicts a schematic representation of integration through singlehomologous recombination.

FIG. 5: Glucoamylase activities of WT 1, WT 2 and transformants ofvarious pGBTOPGLA vectors. Normalized activities are shown, where theactivity of WT 1 at day 4 was set at 100%.

FIG. 6 depicts the plasmid map of pGBDEL-PGGLAA, which is a replacementvector.

FIG. 7 depicts a schematic representation of a promoter replacement.

FIG. 8 depicts a schematic representation of integration throughhomologous recombination.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a promoter DNA sequence selected fromthe group consisting of:

-   -   (a) a DNA sequence comprising the nucleotide sequence of SEQ ID        NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5,    -   (b) a DNA sequence capable of hybridizing with a DNA sequence of        (a),    -   (c) a DNA sequence being at least 50% homologous to a DNA        sequence of (a),    -   (d) a variant of any of the DNA sequences of (a) to (c), and    -   (e) a subsequence of any of the DNA sequences of (a) to (d).

In the context of this invention, a promoter DNA sequence is a DNAsequence, which is capable of controlling the expression of a codingsequence, when this promoter DNA sequence is in operative associationwith this coding sequence. The term “in operative association” isdefined herein as a configuration in which a promoter DNA sequence isappropriately placed at a position relative to a coding sequence suchthat the promoter DNA sequence directs the production of a polypeptideencoded by the coding sequence.

The term “coding sequence” is defined herein as a nucleic acid sequencethat is transcribed into mRNA, which is translated into a polypeptidewhen placed under the control of the appropriate control sequences. Theboundaries of the coding sequence are generally determined by the ATGstart codon, which is normally the start of the open reading frame atthe 5′ end of the mRNA and a transcription terminator sequence locatedjust downstream of the open reading frame at the 3′ end of the mRNA. Acoding sequence can include, but is not limited to, genomic DNA, cDNA,semisynthetic, synthetic, and recombinant nucleic acid sequences.

More specifically, the term “promoter” is defined herein as a DNAsequence that binds the RNA polymerase and directs the polymerase to thecorrect downstream transcriptional start site of a coding sequenceencoding a polypeptide to initiate transcription. RNA polymeraseeffectively catalyzes the assembly of messenger RNA complementary to theappropriate DNA strand of the coding region. The term “promoter” willalso be understood to include the 5′ non-coding region (between promoterand translation start) for translation after transcription into mRNA,cis-acting transcription control elements such as enhancers, and othernucleotide sequences capable of interacting with transcription factors.

In a preferred embodiment, the promoter DNA sequence of the invention isa sequence according to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4 or SEQ ID NO:5.

According to another preferred embodiment, the promoter DNA sequence ofthe invention is a DNA sequence capable of hybridizing with SEQ ID NO 1,SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5, and which stillretains promoter activity.

Promoter activity is preferably determined by measuring theconcentration of the protein(s) coded by the coding sequence(s), whichis (are) in operative association with the promoter. Alternatively thepromoter activity is determined by measuring the enzymatic activity ofthe protein(s) coded by the coding sequence(s), which is (are) inoperative association with the promoter. According to a preferredembodiment, the promoter activity (and its strength) is determined bymeasuring the expression of the coding sequence of the lacZ reportergene (In Luo (Gene 163 (1995) 127-131. According to another preferredembodiment, the promoter activity is determined by using the greenfluorescent protein as coding sequence (In Microbiology. 1999 March; 145(Pt 3):729-34. Santerre Henriksen A L, Even S, Muller C, Punt P J, vanden Hondel C A, Nielsen J. Study).

Additionally, promoter activity can be determined by measuring the mRNAlevels of the transcript generated under control of the promoter. ThemRNA levels can, for example, be measured through a Northern blot (J.Sambrook, E. F. Fritsch, and T. Maniatus, 1989, Molecular Cloning, ALaboratory Manual, 2d edition, Cold Spring Harbor, N.Y.).

The present invention encompasses (isolated) promoter DNA sequences thathybridize under very low stringency conditions, preferably lowstringency conditions, more preferably medium stringency conditions,more preferably medium-high stringency conditions, even more preferablyhigh stringency conditions, and most preferably very high stringencyconditions with a nucleic acid probe that corresponds to (i) nucleotides1 to 2000 of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQID NO:5, preferably nucleotides 100 to 1990, more preferably 200 to1980, even more preferably 300 to 1970, even more preferably 350 to 1950and most preferably 360 to 1900, (ii) is a subsequence of (i), or (iii)is a complementary strand of (i), (ii), (J. Sambrook, E. F. Fritsch, andT. Maniatis, 1989, Molecular Cloning, A Laboratory Manual, 2d edition,Cold Spring Harbor, N.Y.). The subsequence of SEQ ID NO:1, SEQ ID NO:2,SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5, may be at least 100nucleotides, preferably at least 200 nucleotides, more preferably atleast 300 nucleotides, even more preferably at least 400 nucleotides andmost preferably at least 500 nucleotides.

The nucleic acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQID NO:4 or SEQ ID NO:5 or a subsequence thereof may be used to design anucleic acid probe to identify and clone DNA promoters from strains ofdifferent genera or species according to methods well known in the art.In particular, such probes can be used for hybridization with thegenomic or cDNA of the genus or species of interest, following standardSouthern blotting procedures, in order to identify and isolate thecorresponding gene therein. Such probes can be considerably shorter thanthe entire sequence, but should be at least 15, preferably at least 25,and more preferably at least 35 nucleotides in length. Additionally,such probes can be used to amplify DNA promoters though PCR. An exampleof cloning a promoter through PCR is described herein (see example 1.3).Longer probes can also be used. DNA, RNA and Peptide Nucleic Acid (PNA)probes can be used. The probes are typically labelled for detecting thecorresponding gene (for example, with @32 P, @33 P @3 H, @35 S, biotin,or avidin or a fluorescent marker). Such probes are encompassed by thepresent invention.

Thus, a genomic DNA or cDNA library prepared from such other organismsmay be screened for DNA, which hybridizes with the probes describedabove and which encodes a polypeptide. Genomic or other DNA from suchother organisms may be separated by agarose or polyacrylamide gelelectrophoresis, or other separation techniques. DNA from the librariesor the separated DNA may be transferred to and immobilized onnitrocellulose or other suitable carrier material. In order to identifya clone or DNA which is homologous with SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4 or SEQ ID NO:5, or a subsequence thereof, the carriermaterial may be used in a Southern blot.

For purposes of the present invention, hybridization indicates that thenucleic acid sequence hybridizes to a labeled nucleic acid probecorresponding to the nucleic acid sequence shown in SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5, their complementarystrands, or subsequences thereof, under very low to very high stringencyconditions. Molecules to which the nucleic acid probe hybridizes underthese conditions are detected using for example a X-ray film. Otherhybridisation techniques also can be used, such as techniques usingfluorescence for detection and glass sides and/or DNA microarrays assupport. An example of DNA microarray hybridisation detection is givenin FEMS Yeast Res. 2003 December; 4(3):259-69 (Daran-Lapujade P, Daran JM, Kotter P, Petit T, Piper M D, Pronk J T. “Comparative genotyping ofthe Saccharomyces cerevisiae laboratory strains S288C and CEN.PK113-7Dusing oligonucleotide microarrays”. Additionally, the use of PNAmicroarrays for hybridization is described in Nucleic Acids Res. 2003Oct. 1; 31(19):e119 (Brandt O, Feldner J, Stephan A, Schroder M,Schnolzer M, Arlinghaus H F, Hoheisel J D, Jacob A. PNA microarrays forhybridisation of unlabelled DNA samples.)

In a preferred embodiment, the nucleic acid probe is the nucleic acidsequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ IDNO:5. In another preferred embodiment, the nucleic acid probe is thesequence having nucleotides 20 to 1980 of SEQ ID NO:1, SEQ ID NO:2, SEQID NO:3, SEQ ID NO:4 or SEQ ID NO:5, more preferably nucleotides 500 to1950 of SEQ ID NO 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ IDNO:5, even more preferably nucleotides 800 to 1920 of SEQ ID NO 1, SEQID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5, and most preferablynucleotides 900 to 1900 of SEQ ID NO 1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4 or SEQ ID NO:5. Another preferred probe is the part of the DNAsequence before the transcription site.

For long probes of at least 100 nucleotides in length, very low to veryhigh stringency conditions are defined as prehybridization andhybridization at 42 DEG C. in 5.times.SSPE, 0.3% SDS, 200.mu.g/mlsheared and denatured salmon sperm DNA, and either 25% formamide forvery low and low stringencies, 35% formamide for medium and medium-highstringencies, or 50% formamide for high and very high stringencies,following standard Southern blotting procedures.

For long probes of at least 100 nucleotides in length, the carriermaterial is finally washed three times each for 15 minutes using2.times.SSC, 0.2% SDS preferably at least at 45 DEG C. (very lowstringency), more preferably at least at 50 DEG C. (low stringency),more preferably at least at 55 DEG C. (medium stringency), morepreferably at least at 60 DEG C. (medium-high stringency), even morepreferably at least at 65 DEG C. (high stringency), and most preferablyat least at 70 DEG C. (very high stringency).

For short probes which are about 15 nucleotides to about 70 nucleotidesin length, stringency conditions are defined as prehybridization,hybridization, and washing post-hybridization at 5 DEG C. to 10 DEG C.below the calculated Tm using the calculation according to Bolton andMcCarthy (1962, Proceedings of the National Academy of Sciences USA48:1390) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA, 0.5% NP-40,1.times.Denhardt's solution, 1 mM sodium pyrophosphate, 1 mM sodiummonobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per mlfollowing standard Southern blotting procedures.

For short probes which are about 15 nucleotides to about 70 nucleotidesin length, the carrier material is washed once in 6.times.SCC plus 0.1%SDS for 15 minutes and twice each for 15 minutes using 6.times.SSC at 5DEG C. to 10 DEG C. below the calculated Tm.

According to another preferred embodiment, SEQ ID NO 1, SEQ ID NO:2, SEQID NO:3, SEQ ID NO:4 or SEQ ID NO:5 is first used to clone the nativegene, coding sequence or part of it, which is operatively associatedwith it. This can be done starting with either SEQ ID NO 1, SEQ ID NO:2,SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5, or a subsequence thereof asearlier defined and using this sequence as a probe. The probe ishybridised to a cDNA or a genomic library of a given host, eitherAspergillus niger or any other fungal host as defined in thisapplication. Once the native gene or part of it has been cloned, it canbe subsequently used itself as a probe to clone homologous genes thereofderived from other fungi by hybridisation experiments as describedherein.

In the context of the invention, a homologous gene means a gene, whichis at least 50% homologous (identical) to the native gene. Preferably,the homologous gene is at least 55% homologous, more preferably at least60%, more preferably at least 65%, more preferably at least 70%, evenmore preferably at least 75% preferably about 80%, more preferably about90%, even more preferably about 95%, and most preferably about 97%homologous to the native gene.

The sequence upstream of the coding sequence of the homologous gene is apromoter encompassed by the present invention. Alternatively, thesequence of the native gene, coding sequence or part of it, which isoperatively associated with a promoter of the invention can beidentified by using SEQ ID NO 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4or SEQ ID NO:5 or a subsequence thereof as earlier defined to searchgenomic databases using for example an alignment or BLAST algorithm asdescribed herein. This identified sequence subsequently can be used toidentify orthologues or homologous genes in any other fungal host asdefined in this application. The sequence upstream the coding sequenceof the identified orthologue or homologous gene is a promoterencompassed by the present invention.

According to another preferred embodiment, the promoter DNA sequence ofthe invention is a(n) (isolated) DNA sequence, which is at least 50%homologous (identical) to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4 or SEQ ID NO:5. Preferably, the DNA sequence is at least 55%homologous, more preferably at least 60%, more preferably at least 65%,more preferably at least 70%, even more preferably at least 75%preferably about 80%, more preferably about 90%, even more preferablyabout 95%, and most preferably about 97% homologous to SEQ ID NO:1, SEQID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.

For purposes of the present invention, the degree of homology (identity)between two nucleic acid sequences is preferably determined by the BLASTprogram. Software for performing BLAST analyses is publicly availablethrough the National Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). The BLAST algorithm parameters W, T, andX determine the sensitivity and speed of the alignment. The BLASTprogram uses as defaults a wordlength (W) of 11, the BLOSUM62 scoringmatrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915(1989)) alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and acomparison of both strands.

In another preferred embodiment, the promoter is a subsequence of SEQ IDNO 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5, thesubsequence still having promoter activity. The subsequence preferablycontains at least about 100 nucleotides, more preferably at least about200 nucleotides, and most preferably at least about 300 nucleotides.

In another preferred embodiment, a subsequence is a nucleic acidsequence encompassed by SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4 or SEQ ID NO:5 except that one or more nucleotides from the 5′and/or 3′ end have been deleted, said DNA sequence still having promoteractivity.

In another preferred embodiment, the promoter subsequence is a ‘trimmed’subsequence, i.e. a sequence fragment which is upstream from translationstart and/or from transcription start. An example of trimming a promoterand functionally analysing it is described in Gene. 1994 Aug. 5;145(2):179-87: the effect of multiple copies of the upstream region onexpression of the Aspergillus nicer glucoamylase-encoding gene. VerdoesJ C, Punt P J, Stouthamer A H, van den Hondel C A).

In another embodiment of the invention, the promoter DNA sequence is avariant of SEQ ID NO 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ IDNO:5.

The term “variant” or “variant promoter” is defined herein as a promoterhaving a nucleotide sequence comprising a substitution, deletion, and/orinsertion of one or more nucleotides of a parent promoter, wherein thevariant promoter has more or less promoter activity than thecorresponding parent promoter. The term “variant promoter” willencompass natural variants and in vitro generated variants obtainedusing methods well known in the art such as classical mutagenesis,site-directed mutagenesis, and DNA shuffling. A variant promoter mayhave one or more mutations. Each mutation is an independentsubstitution, deletion, and/or insertion of a nucleotide.

According to a preferred embodiment, the variant promoter is a promoter,which has at least a modified regulatory site as compared to thepromoter sequence first identified (SEQ ID NO 1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4 or SEQ ID NO:5). Such a regulatory site can be removedin its entirety or specifically mutated as explained above. Theregulation of such promoter variant is thus modified so that for exampleit is no longer induced by glucose. Examples of such promoter variantsand techniques on how to obtain them are described in EP 673 429 or inWO 94/04673. These patents are herewith incorporated by reference.

The promoter variant can be an allelic variant. An allelic variantdenotes any of two or more alternative forms of a gene occupying thesame chromosomal locus. Allelic variation arises naturally throughmutation, and may result in polymorphism within populations. The variantpromoter may be obtained by (a) hybridizing a DNA under very low, low,medium, medium-high, high, or very high stringency conditions with (i)SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5, (ii)a subsequence of (i) or (iii) a complementary strand of (i), (ii), and(b) isolating the variant promoter from the DNA. Stringency and washconditions are as defined herein.

The promoter of the invention can be a promoter, whose sequence may beprovided with linkers for the purpose of introducing specificrestriction sites facilitating ligation of the promoter sequence withthe coding region of the nucleic acid sequence encoding a polypeptide.

The sequence information as provided herein should not be so narrowlyconstrued as to require inclusion of erroneously identified bases. Thespecific sequences disclosed herein can readily be used to isolate theoriginal DNA sequence from a filamentous fungus, in particularAspergillus niger, and be subjected to further sequence analyses therebyidentifying sequencing errors.

Unless otherwise indicated, all nucleotide sequences determined bysequencing a DNA molecule herein were determined using an automated DNAsequencer. Therefore, as is known in the art for any DNA sequencedetermined by this automated approach, any nucleotide sequencedetermined herein may contain some errors. Nucleotide sequencesdetermined by automation are typically at least about 90% identical,more typically at least about 95% to at least about 99.9% identical tothe actual nucleotide sequence of the sequenced DNA molecule. The actualsequence can be more precisely determined by other approaches includingmanual DNA sequencing methods well known in the art.

The person skilled in the art is capable of identifying such erroneouslyidentified bases and knows how to correct for such errors.

The present invention encompasses functional promoter equivalentstypically containing mutations that do not alter the biological functionof the promoter it concerns. The term “functional equivalents” alsoencompasses orthologues of the A. niger DNA sequences. Orthologues ofthe A. niger DNA sequences are DNA sequences that can be isolated fromother strains or species and possess a similar or identical biologicalactivity.

The promoter sequences of the present invention may be obtained frommicroorganisms of any genus. For purposes of the present invention, theterm “obtained from” as used herein in connection with a given sourceshall mean that the polypeptide is produced by the source or by a cellin which a gene from the source has been inserted.

The promoter sequences may be obtained from a fungal source, preferablyfrom a yeast strain such as a Candida, Hansenula, Kluyveromyces, Pichia,Saccharomyces, Schizosaccharomyces, or Yarrowia strain, more preferablyfrom a Saccharomyces carlsbergensis, Saccharomyces cerevisiae,Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyceskluyveri, Saccharomyces norbensis or so Saccharomyces oviformis strain.

In another preferred embodiment, the promoter sequences are obtainedfrom a filamentous fungal strain such as an Acremonium, Aspergillus,Aureobasidium, Cryptococcus, Filibasidium, Fusarium, Humicola,Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora,Paecilomyces, Penicillium, Piromyces, Schizophyllum, Talaromyces,Thermoascus, Thielavia, Tolypocladium, or Trichoderma strain, morepreferably from an Aspergillus aculeatus, Aspergillus awamori,Aspergillus foetidus, Aspergillus japonicus, A. nidulans, A. niger,Aspergillus oryzae (A. oryzae), Humicola insolens, Humicola lanuginosa,Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicilliumpurpurogenum, Trichoderma harzianum, Trichoderma koningii, Trichodermalongibrachiatum, Trichoderma reesei, or Trichoderma viride strain.

In another preferred embodiment, the promoter sequences are obtainedfrom a Fusarium bactridioides, Fusarium cerealis, Fusariumcrookwellense, Fusarium culmorum, Fusarium graminearum, Fusariumgraminum, Fusarium heterosporum Fusarium negundi, Fusarium oxysporum,Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusariumsarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusariumtorulosum, Fusarium trichothecioides, Fusarium venenatum strain.

It will be understood that for the aforementioned species, the inventionencompasses the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents. Strains of these species arereadily accessible to the public in a number of culture collections,such as the American Type Culture Collection (ATCC), Deutsche Sammlungvon Mikroorganismen and Zellkulturen GmbH (DSM), Centraalbureau VoorSchimmelcultures (CBS), and Agricultural Research Service Patent CultureCollection, Northern Regional Research Center (NRRL).

Furthermore, promoter sequences according to the invention may beidentified and obtained from other sources including microorganismsisolated from nature (e.g, soil, composts, water, etc.) using theabove-mentioned probes. Techniques for isolating microorganisms fromnatural habitats are well known in the art. The nucleic acid sequencemay then be derived by similarly screening a genomic DNA library ofanother microorganism. Once a nucleic acid sequence encoding a promoterhas been detected with the probe(s), the sequence may be isolated orcloned by utilizing techniques which are known to those of ordinaryskill in the art (see, e.g., Sambrook et al., 1989, supra).

In the present invention, the promoter DNA sequence may also be a hybridpromoter comprising a portion of one or more promoters of the presentinvention; a portion of a promoter of the present invention and aportion of another known promoter, e.g., a leader sequence of onepromoter and the transcription start site from the other promoter; or aportion of one or more promoters of the present invention and a portionof one or more other promoters. The other promoter may be any promotersequence, which shows transcriptional activity in the fungal host cellof choice including a variant, truncated, and hybrid promoter, and maybe obtained from genes encoding extracellular or intracellularpolypeptides either homologous or heterologous to the host cell. Theother promoter sequence may be native or foreign to the nucleic acidsequence encoding the polypeptide and native or foreign to the cell.

As a preferred embodiment, important regulatory subsequences of thepromoter identified can be fused to other ‘basic’ promoters to enhancetheir promoter activity (as for example described in Mol. Microbiol.1994 May; 12(3):479-90. Regulation of the xylanase-encoding xlnA gene ofAspergillus tubigensis. de Graaff L H, van den Broeck H C, van Ooijen AJ, Visser J.).

Other examples of other promoters useful in the construction of hybridpromoters with the promoters of the present invention include thepromoters obtained from the genes for A. oryzae TAKA amylase, Rhizomucormiehei esparto proteinase, A. niger neutral alpha-amylase, A. niger acidstable alpha-amylase, A. niger or Aspergillus awamori glucoamylase(glaA), A. niger gpdA, A. niger glucose oxidase goxC, Rhizomucor mieheilipase, A. oryzae alkaline protease, A. oryzae triose phosphateisomerase, A. nidulans acetamidase, and Fusarium.oxysporum trypsin-likeprotease (WO 96/00787), as well as the NA2-tpi promoter (a hybrid of thepromoters from the genes for A. niger neutral alpha-amylase and A.oryzae triose phosphate isomerase), Saccharomyces cerevisiae enolase(ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomycescerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphatedehydrogenase (ADH2/GAP), and Saccharomyces cerevisiae3-phosphoglycerate kinase, and mutant, truncated, and hybrid promotersthereof. Other useful promoters for yeast host cells are described byRomanoset al., 1992, Yeast 8: 423-488.

In the present invention, the promoter DNA sequence may also be a“tandem promoter”. A “tandem promoter” is defined herein as two or morepromoter sequences each of which is in operative association with acoding sequence and mediates the transcription of the coding sequenceinto mRNA.

The tandem promoter comprises two or more promoters of the presentinvention or alternatively one or more promoters of the presentinvention and one or more other known promoters, such as thoseexemplified above useful for the construction of hybrid promoters. Thetwo or more promoter sequences of the tandem promoter may simultaneouslypromote the transcription of the nucleic acid sequence. Alternatively,one or more of the promoter sequences of the tandem promoter may promotethe transcription of the nucleic acid sequence at different stages ofgrowth of the cell or morphological different parts of the mycelia.

In the present invention, the promoter may be foreign to the codingsequence encoding a polypeptide of interest and/or to the fungal hostcell. A variant, hybrid, or tandem promoter of the present inventionwill be understood to be foreign to a coding sequence encoding apolypeptide even if the wild-type promoter is native to the codingsequence or to the fungal host cell.

A variant, hybrid, or tandem promoter of the present invention has atleast about 20%, preferably at least about 40%, more preferably at leastabout 60%, more preferably at least about 80%, more preferably at leastabout 90%, more preferably at least about 100%, even more preferably atleast about 200%, most preferably at least about 300%, and even mostpreferably at least about 400% of the promoter activity of the promoterhaving SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ IDNO:5.

The invention further relates to a DNA construct comprising at least onepromoter DNA sequence as defined above and a coding sequence inoperative association with said promoter DNA sequence such that thecoding sequence can be expressed under the control of the promoter DNAsequence in a given fungal host cell.

The coding sequence encodes a polypeptide that may be native orheterologous to the fungal host cell of interest.

The term “polypeptide” is not meant herein to refer to a specific lengthof the encoded product and, therefore, encompasses peptides,oligopeptides, and proteins. The term “heterologous polypeptide” isdefined herein as a polypeptide, which is not native to the fungal cell,a native polypeptide in which modifications have been made to alter thenative sequence, or a native polypeptide whose expression isquantitatively altered as a result of a manipulation of the fungal cellby recombinant DNA techniques. For example, a native polypeptide may berecombinantly produced by, e.g., placing the sequence encoding thepolypeptide under the control of the promoter of the present inventionto enhance expression of the polypeptide, to expedite export of a nativepolypeptide of interest outside the cell by use of a signal sequence,and to increase the copy number of a gene encoding the polypeptidenormally produced by the cell. The fungal cell may contain one or morecopies of the coding sequence encoding the polypeptide.

Preferably, the coding sequence encodes a peptide hormone or variantthereof, an enzyme, an intracellular protein, a protein involved insecretion process, a protein involved in folding process, a chaperone, apeptide amino acid transporter, a glycosylation factor, a transcriptionfactor, a receptor or portion thereof, an antibody or portion thereof,or a reporter protein.

In a preferred embodiment, the polypeptide is secreted extracellularly.In a more preferred embodiment, the polypeptide is an enzyme. Examplesof enzymes are cellulases such as endoglucanases, β-glucanases,cellobiohydrolases or β-glucosidases; hemicellulases or pectinolyticenzymes such as xylanases, xylosidases, mannanases, galactanases,galactosidases, pectin methyl esterases, pectin lyases, pectate lyases,endo polygalacturonases, exopolygalacturonases rhamnogalacturonases,arabanases, arabinofuranosidases, arabinoxylan hydrolases,galacturonases, lyases; amylolytic enzymes; phosphatases such asphytases, esterase such as lipases, proteolytic enzyme, such asproteases, peptidases, oxidoreductases such as oxidases, transferases,or isomerases.

Alternatively, the coding sequence may encode an intracellular proteinsuch as for example a chaperone or transcription factor. An example ofthis is described in Appl Microbiol Biotechnol. 1998 October;50(4):447-54 (“Analysis of the role of the gene bipA, encoding the majorendoplasmic reticulum chaperone protein in the secretion of homologousand heterologous proteins in black Aspergilli. Punt P J, van Gemeren IA, Drint-Kuijvenhoven J, Hessing J G, van Muijlwijk-Harteveld G M,Beijersbergen A, Verrips C T, van den Hondel C A). This can be used forexample to improve the efficiency of a host cell as protein producer ifthis coding sequence, such as a chaperone or transcription factor, wasknown to be a limiting factor in protein production.

The coding sequence may also encode an enzyme involved in the synthesisof a primary or secondary metabolite, such as organic acids,carotenoids, (beta-lactam) antibiotics, vitamins.

The coding sequence encoding a polypeptide of interest may be obtainedfrom any prokaryotic, eukaryotic, or other source.

Alternatively, the coding sequence may code for the expression of anantisense RNA and/or an RNAi (RNA interference) construct. An example ofexpressing an antisense-RNA is shown in Appl Environ Microbiol. 2000February; 66(2):775-82. (Characterization of a foldase, proteindisulfide isomerase A, in the protein secretory pathway of Aspergillusniger. Ngiam C, Jeenes D J, Punt P J, Van Den Hondel C A, Archer D B) or(Zrenner R, Willmitzer L, Sonnewald U. Analysis of the expression ofpotato uridinediphosphate-glucose pyrophosphorylase and its inhibitionby antisense RNA. Planta. (1993);190(2):247-52.) Complete inactivationof the expression of a gene is useful for instance for the inactivationof genes controlling undesired side branches of metabolic pathways, forinstance to increase the production of specific secondary metabolitessuch as (beta-lactam) antibiotics or carotenoids. Complete inactivationis also useful to reduce the production of toxic or unwanted compounds(chrysogenin in Penicillium; Aflatoxin in Aspergillus: MacDonald K D etal,: heterokaryon studies and the genetic control of penicillin andchrysogenin production in Penicillium chrysogenum. J Gen Microbiol.(1963) 33:375-83). Complete inactivation is also useful to alter themorphology of the organism in such a way that the fermentation processand down stream processing is improved.

Another embodiment of the invention relates to the extensive metabolicreprogramming or engineering of a fungal cell. Introduction of completenew pathways and/or modification of unwanted pathways will provide acell specifically adapted for the production of a specific compound suchas a protein or a metabolite.

In the methods of the present invention, when the coding sequence codesfor a polypeptide, said polypeptide may also include a fused or hybridpolypeptide in which another polypeptide is fused at the N-terminus orthe C-terminus of the polypeptide or fragment thereof. A fusedpolypeptide is produced by fusing a nucleic acid sequence (or a portionthereof) encoding one polypeptide to a nucleic acid sequence (or aportion thereof) encoding another polypeptide. Techniques for producingfusion polypeptides are known in the art, and include, ligating thecoding sequences encoding the polypeptides so that they are in frame andexpression of the fused polypeptide is under control of the samepromoter(s) and terminator. The hybrid polypeptide may comprise acombination of partial or complete polypeptide sequences obtained fromat least two different polypeptides wherein one or more may beheterologous to the fungal cell.

The DNA construct may comprise one or more control sequences in additionto the promoter DNA sequence, which direct the expression of the codingsequence in a suitable host cell under conditions compatible with thecontrol sequences. Expression will be understood to include any stepinvolved in the production of the polypeptide including, but not limitedto, transcription, post-transcriptional modification, translation,post-translational modification, and secretion. One or more controlsequences may be native to the coding sequence or to the host.Alternatively, one or more control sequences may be replaced with one ormore control sequences foreign to the nucleic acid sequence forimproving expression of the coding sequence in a host cell.

“DNA construct” is defined herein as a nucleic acid molecule, eithersingle or double-stranded, which is isolated from a naturally occurringgene or which has been modified to contain segments of nucleic acidcombined and juxtaposed in a manner that would not otherwise exist innature. The term DNA construct is synonymous with the term expressioncassette when the DNA construct contains a coding sequence and all thecontrol sequences required for expression of the coding sequence.

The term “control sequences” is defined herein to include allcomponents, which are necessary or advantageous for the expression of acoding sequence, including the promoter of the invention. Each controlsequence may be native or foreign to the nucleic acid sequence encodingthe polypeptide. Such control sequences include, but are not limited to,a leader, an optimal translation initiation sequence (as described inKozak, 1991, J. Biol. Chem. 266:19867-19870), a polyadenylationsequence, a propeptide sequence, a signal peptide sequence, an upstreamactivating sequence, the promoter of the invention including variants,fragments, and hybrid and tandem promoters derived thereof and atranscription terminator. At a minimum, the control sequences includetranscriptional and translational stop signals and (part of) thepromoter of the invention. The control sequences may be provided withlinkers for the purpose of introducing specific restriction sitesfacilitating ligation of the control sequences with the coding region ofthe nucleic acid sequence encoding a polypeptide.

The control sequence may be a suitable transcription terminatorsequence, i.e. a sequence recognized by a host cell to terminatetranscription. The terminator sequence is in operative association withthe 3′ terminus of the coding sequence encoding the polypeptide. Anyterminator, which is functional in the host cell of choice may be usedin the present invention.

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for A. oryzae TAKA amylase, A. niger glucoamylase, A.nidulans anthranilate synthase, A. niger alpha-glucosidase, trpC gene,and Fusarium oxysporum trypsin-like protease.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C(CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al, 1992, supra.

The control sequence may also be a suitable leader sequence, i.e. a 5′nontranslated region of a mRNA which is important for translation by thehost cell. The leader sequence is in operative association with the 5′terminus of the nucleic acid sequence encoding the polypeptide. Anyleader sequence that is functional in the host cell of choice may beused in the present invention.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for A. oryzae TAKA amylase, A. nidulans triosephosphateisomerase and A. niger glaA.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequencein operative association with the 3′ terminus of the nucleic acidsequence and which, when transcribed, is recognized by the host cell asa signal to add polyadenosine residues to transcribed mRNA. Anypolyadenylation sequence, which is functional in the host cell of choicemay be used in the present invention.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for A. oryzae TAKA amylase, A. nigerglucoamylase, A. nidulans anthranilate synthase, Fusarium oxysporumtrypsin-like protease, and A. niger alpha-glucosidase.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Molecular Cellular Biology 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatcodes for an amino acid sequence linked to the amino terminus of apolypeptide and directs the encoded polypeptide into the cell'ssecretory pathway. The 5′ end of the coding sequence of the nucleic acidsequence may inherently contain a signal peptide coding region naturallylinked in translation reading frame with the segment of the codingregion which encodes the secreted polypeptide. Alternatively, the 5′ endof the coding sequence may contain a signal peptide coding region whichis foreign to the coding sequence. The foreign signal peptide codingregion may be required where the coding sequence does not naturallycontain a signal peptide coding region. Alternatively, the foreignsignal peptide coding region may simply replace the natural signalpeptide coding region in order to enhance secretion of the polypeptide.However, any signal peptide coding region which directs the expressedpolypeptide into the secretory pathway of a host cell of choice may beused in the present invention.

Effective signal peptide coding regions for filamentous fungal hostcells are the signal peptide coding regions obtained from the genes forA. oryzae TAKA amylase, A. niger neutral amylase, A. ficuum phytase, A.niger glucoamylase, A. niger endoxylanase, Rhizomucor miehei asparticproteinase, Humicola insolens cellulase, and Humicola lanuginosa lipase.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding regions are described byRomanoset al., 1992, supra.

The control sequence may also be a propeptide coding region that codesfor an amino acid sequence positioned at the amino terminus of apolypeptide. The resultant polypeptide is known as a proenzyme orpropolypeptide (or a zymogen in some cases). A propolypeptide isgenerally inactive and can be converted to a mature active polypeptideby catalytic or autocatalytic cleavage of the propeptide from thepropolypeptide. The propeptide coding region may be obtained from thegenes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilisneutral protease (nprT), Saccharomyces cerevisiae alpha-factor,Rhizomucor miehei aspartic proteinase, Myceliophthora thermophilalaccase (WO 95/33836) and A. niger endoxylanase (endo1).

Where both signal peptide and propeptide regions are present at theamino terminus of a polypeptide, the propeptide region is positionednext to the amino terminus of a polypeptide and the signal peptideregion is positioned next to the amino terminus of the propeptideregion.

It may also be desirable to add regulatory sequences, which allow theregulation of the expression of the polypeptide relative to the growthof the host cell. Examples of regulatory systems are those which causethe expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. Regulatory systems in prokaryotic systems include the lac, andtrp operator systems. In yeast, the ADH2 system or GAL1 system may beused. In filamentous fungi, the TAKA alpha-amylase promoter, A. nigerglucoamylase promoter, A. oryzae glucoamylase promoter, A. tubingensisendoxylanase (xlnA) promoter, A. niger nitrate reductase (niaD)promoter, Trichoderma reesei cellobiohydrolase promoter and the A.nidulans alcohol and aldehyde dehydrogenase (alcA and aldA,respectively) promoters as described in U.S. Pat. No. 5,503,991) may beused as regulatory sequences. Other examples of regulatory sequences arethose, which allow for gene amplification. In eukaryotic systems, theseinclude the dihydrofolate reductase gene, which is amplified in thepresence of methotrexate, and the metallothionein genes, which areamplified with heavy metals. In these cases, the nucleic acid sequenceencoding the polypeptide would be in operative association with theregulatory sequence.

Important can be removal of creA binding sites (carbon cataboliterepression as described earlier in EP 673 429), change of pacC and areA(for pH and nitrogen regulation).

The present invention also relates to recombinant expression vectorscomprising a promoter of the present invention, a coding sequenceencoding a polypeptide, and transcriptional and translational stopsignals. The various coding and control sequences described above may bejoined together to produce a recombinant expression vector which mayinclude one or more convenient restriction sites to allow for insertionor substitution of the promoter and/or coding sequence encoding thepolypeptide at such sites: Alternatively, fusion of coding sequence andpromoter can be done by e.g. sequence, overlap extension using PCR(SOE-PCR), as described in Gene. 1989 Apr. 15; 77(1):51-9. Ho S N, HuntH D, Horton R M, Pullen J K, Pease L R “Site-directed mutagenesis byoverlap extension using the polymerase chain reaction”) or by cloningusing the Gateway™ cloning system (Invitrogen). Alternatively, thecoding sequence may be expressed by inserting the coding sequence or aDNA construct comprising the promoter and/or coding sequence into anappropriate vector for expression. In creating the expression vector,the coding sequence is located in the vector so that the coding sequenceis in operative association with a promoter of the present invention andone or more appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus), which can be conveniently subjected to recombinant DNAprocedures and can bring about the expression of the coding sequence.The choice of the vector will typically depend on the compatibility ofthe vector with the host cell into which the vector is to be introduced.The vectors may be linear or closed circular plasmids.

The vector may be an autonomously replicating vector, i.e., a vector,which exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.For autonomous replication, the vector may comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. Examples of origins of replication for use in a yeasthost cell are the 2 micron origin of replication, ARS1, ARS4, thecombination of ARS1 and CEN3, and the combination of ARS4 and CEN6. Theorigin of replication may be one having a mutation which makes itsfunctioning temperature-sensitive in the host cell (see, e.g., Ehrlich,1978, Proceedings of the National Academy of Sciences USA 75:1433). Anexample of an autonomously maintained cloning vector in a filamentousfungus is a cloning vector comprising the AMA1-sequence. AMA1 is a6.0-kb genomic DNA fragment isolated from A. nidulans, which is capableof Autonomous Maintenance in Aspergillus (see e.g. Aleksenko andClutterbuck (1997), Fungal Genet. Biol. 21: 373-397).

Alternatively, the vector may be one which, when introduced into thehost cell, is integrated into the genome and replicated together withthe chromosome(s) into which it has been integrated. Furthermore, asingle vector or plasmid or two or more vectors or plasmids whichtogether contain the total DNA to be introduced into the genome of thehost cell, or a transposon may be used.

The vectors of the present invention preferably contain one or moreselectable markers, which permit easy selection of transformed cells.The host may be co-transformed with at least two vectors, one comprisingthe selection marker. A selectable marker is a gene the product of whichprovides for biocide or viral resistance, resistance to heavy metals,prototrophy to auxotrophs, and the like. Suitable markers for yeast hostcells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectablemarkers for use in a filamentous fungal host cell include, but are notlimited to, amdS (acetamidase), argB (ornithine carbamoyltransferase),bar (phosphinothricin acetyltransferase), hygB (hygromycinphosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),trpC (anthranilate synthase), as well as equivalents thereof. Markerconferring resistance against e.g. phleomycin, hygromycin B or G418 canalso be used. Preferred for use in an Aspergillus cell are the amdS andpyrG genes of A. nidulans or A. oryzae and the bar gene of Streptomyceshygroscopicus. The amdS marker gene is preferably used applying thetechnique described in EP 635 574 or WO 97/0626. A preferred selectionmarker gene is the A. nidulans amdS coding sequence fused to the A.nidulans gpdA promoter (EP635 574). AmdS genes from other filamentousfungus may also be used (WO 97/06261).

For integration into the host cell genome, the vector may rely on thepromoter sequence and/or coding sequence encoding the polypeptide or anyother element of the vector for stable integration of the vector intothe genome by homologous or non-homologous recombination. Alternatively,the vector may contain additional nucleic acid sequences for directingintegration by homologous recombination into the genome of the hostcell. The additional nucleic acid sequences enable the vector to beintegrated into the host cell genome at a precise location(s) in thechromosome(s). To increase the likelihood of integration at a preciselocation, the integrational elements should preferably contain asufficient number of nucleic acids, such as 100 to 1,500 base pairs,preferably 400 to 1,500 base pairs, more preferably 800 to 1,500 basepairs, and most preferably at least 2 kb, which are highly homologouswith the corresponding target sequence to enhance the probability ofhomologous recombination. The integrational elements may be any sequencethat is homologous with the target sequence in the genome of the hostcell. Furthermore, the integrational elements may be non-encoding orencoding nucleic acid sequences. In order to promote targetedintegration, the cloning vector is preferably linearized prior totransformation of the host cell. Linearization is preferably performedsuch that at least one but preferably either end of the cloning vectoris flanked by sequences homologous to the target locus.

Preferably, the integrational elements in the cloning vector, which arehomologous to the target locus are derived from a highly expressed locusmeaning that they are derived from a gene, which is capable of highexpression level in the fungal host cell. A gene capable of highexpression level, i.e. a highly expressed gene, is herein defined as agene whose mRNA can make up at least 0.5% (w/w) of the total cellularmRNA, e.g. under induced conditions, or alternatively, a gene whose geneproduct can make up at least 1% (w/w) of the total cellular protein, or,in case of a secreted gene product, can be secreted to a level of atleast 0.1 g/l (as described in EP 357 127 B1). A number of preferredhighly expressed fungal genes are given by way of example: the amylase,glucoamylase, alcohol dehydrogenase, xylanase, glyceraldehyde-phosphatedehydrogenase or cellobiohydrolase genes from Aspergilli or Trichoderma.Most preferred highly expressed genes for these purposes are aglucoamylase gene, preferably an A. niger glucoamylase gene, an A.oryzae TAKA-amylase gene, an A. nidulans gpdA gene, the loci of SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5, the A. nigerlocus of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ IDNO:5, or a Trichoderma reesei cellobiohydrolase gene.

On the other hand, the vector may be integrated into the genome of thehost cell by non-homologous recombination.

More than one copy of a nucleic acid sequence encoding a polypeptide maybe inserted into the host cell to increase production of the geneproduct. This can be done, preferably by integrating into its genomecopies of the DNA sequence, more preferably by targeting the integrationof the DNA sequence at a highly expressed locus, preferably at aglucoamylase locus or at the locus of SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4 or SEQ ID NO:5. Alternatively, this can be done byincluding an amplifiable selectable marker gene with the nucleic acidsequence where cells containing amplified copies of the selectablemarker gene, and thereby additional copies of the nucleic acid sequence,can be selected for by cultivating the cells in the presence of theappropriate selectable agent. To increase even more the number of copiesof the DNA sequence to be over expressed the technique of geneconversion as described in WO98/46772 may be used.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

The present invention also relates to recombinant host cells, comprisinga promoter DNA sequence of the present invention in operativeassociation with a coding sequence encoding a polypeptide, which areadvantageously used in the production of the polypeptides. A vectorcomprising a promoter of the present invention in operative associationwith a coding sequence encoding a polypeptide is introduced into a hostcell so that the vector is maintained as a chromosomal integrant or as aself-replicating extra-chromosomal vector as described earlier. The term“host cell” encompasses any progeny of a parent cell that is notidentical to the parent cell due to mutations that occur duringreplication. The choice of a host cell will to a large extent dependupon the gene encoding the polypeptide and its source.

The present invention also relates to recombinant host cells, comprisingmore than one promoter DNA sequence of the present invention, eachpromoter being in operative association with a coding sequence encodinga polypeptide. Such host cells may be advantageously used in therecombinant production of at least one polypeptide. Preferably at leastone promoter and its associated coding sequence are present on a vector.The vector is introduced into a host cell so that it is maintained as achromosomal integrant and/or as a self-replicating extra-chromosomalvector as described earlier.

According to another preferred embodiment, the host cell is used for theproduction of specific primary or secondary metabolites such as(beta-lactam) antibiotics, vitamins or carotenoids.

The host cell may be any fungal cell useful in the methods of thepresent invention. “Fungi” as used herein includes the phyla Ascomycota,Basidiomycota, Chytridiomycota, and Zygomycota (as defined by Hawksworthet al., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition,1995, CAB International, University Press, Cambridge, UK) as well as theOomycota (as cited in Hawksworth et al., 1995, supra, page 171) and allmitosporic fungi (Hawksworth et al., 1995, supra).

In a preferred embodiment, the fungal host cell is a yeast cell. “Yeast”as used herein includes ascosporogenous yeast (Endomycetales),basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti(Blastomycetes). Since the classification of yeast may change in thefuture, for the purposes of this invention, yeast shall be defined asdescribed in Biology and Activities of Yeast (Skinner, F. A., Passmore,S. M., and Davenport, R. R., eds, Soc. App. Bacteriol. Symposium SeriesNo. 9, 1980).

In a more preferred embodiment, the yeast host cell is a Candida,Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, orYarrowia cell.

In a most preferred embodiment, the yeast host cell is a Saccharomycescarlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus,Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensisor Saccharomyces oviformis cell. In another most preferred embodiment,the yeast host cell is a Kluyveromyces lactis cell. In another mostpreferred embodiment, the yeast host cell is a Yarrowia lipolytica cell.

In another preferred embodiment, the fungal host cell is a filamentousfungal cell. “Filamentous fungi” include all filamentous forms of thesubdivision Eumycota and Oomycota (as defined by Hawksworth et al.,1995, supra). The filamentous fungi are characterized by a mycelial wallcomposed of chitin, cellulose, glucan, chitosan, mannan, and othercomplex polysaccharides. Vegetative growth is by hyphal elongation andcarbon catabolism is obligatory aerobic. In contrast, vegetative growthby yeasts such as Saccharomyces cerevisiae is by budding of aunicellular thallus and carbon catabolism may be fermentative.

In a more preferred embodiment, the filamentous fungal host cell is acell of a species of, but not limited to, Acremonium, Aspergillus,Fusarium, Humicola, Mucor, Myceliophthora, Neurospora, Penicillium,Thielavia, Tolypocladium, or Trichoderma.

In a most preferred embodiment, the filamentous fungal host cell is anAspergillus awamori, Aspergillus foetidus, Aspergillus japonicus, A.nidulans, A. niger or A. oryzae cell. In another most preferredembodiment, the filamentous fungal host cell is a Fusariumbactridioides, Fusarium cerealis, Fusarium crookwellense, Fusariumculmorum, Fusarium graminearum, Fusarium graminum, Fusariumheterosporum, Fusarium negundi, Fusarium oxysporum, Fusariumreticulatun, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum,Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum,Fusarium trichothecioides, or Fusarium venenatum cell. In another mostpreferred embodiment, the filamentous fungal host cell is a Humicolainsolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila,Neurospora crassa, Penicillium purpurogenum, Thielavia terrestris,Trichoderma harzianum, Trichoderma koningii, Trichodermalongibrachiatum, Trichoderma reesei, or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus host cells are described in EP 238 023 andYelton et al., 1984, Proceedings of the National Academy of Sciences USA81: 1470-1474. Suitable procedures for transformation of Aspergillus andother filamentous fungal host cells using Agrobacterium tumefaciens aredescribed in e.g. Nat. Biotechnol. 1998 September; 16(9):839-42. Erratumin: Nat Biotechnol 1998 November; 16(11):1074. Agrobacteriumtumefaciens-mediated transformation of filamentous fungi. de Groot M J,Bundock P, Hooykaas P J, Beijersbergen A G. Unilever Research LaboratoryVlaardingen, The Netherlands. Suitable methods for transforming Fusariumspecies are described by Malardier et al., 1989, Gene 78: 147-156 and WO96/00787. Yeast may be transformed using the procedures described byBecker and Guarente, In Abelson, J. N. and Simon, M. I., editors, Guideto Yeast Genetics and Molecular Biology, Methods in Enzymology, Volume194, pp 182-187, Academic Press, Inc., New York; Ito et al., 1983,Journal of Bacteriology 153: 163; and Hinnen et al., 1978, Proceedingsof the National Academy of Sciences USA 75: 1920.

The invention further relates to a method for expression of a codingsequence in a host cell. The method comprises the following steps:

-   -   (a) providing a DNA construct comprising the promoter DNA        sequence of the invention and the coding sequence as described        above,    -   (b) transforming a suitable host cell with said DNA construct        and    -   (c) expressing the coding sequence under the control of said        promoter DNA sequence.

The invention also relates to a method for production of a polypeptideencoded by a coding sequence that is under control of the promoter ofthe invention in a suitable fungal host. The method comprises thefollowing steps:

-   -   (a) providing a DNA construct comprising the promoter DNA        sequence of the invention and the coding sequence encoding the        polypeptide as defined above,    -   (b) transforming a suitable fungal host cell with said DNA        construct,    -   (c) culturing the suitable fungal host under suitable culture        conditions conducive to expression of the polypeptide,    -   (d) recovering the polypeptide from the culture broth.

The invention also relates to a method for production of a secondarymetabolite in a suitable host comprising:

-   -   (a) providing a DNA construct comprising the promoter DNA        sequence of the invention and a coding sequence encoding an        enzyme involved in the production of a secondary metabolite as        described above,    -   (b) transforming a suitable host cell with said DNA construct,    -   (c) culturing the suitable fungal host under suitable culture        conditions conducive to production of the secondary metabolite,        and    -   (d) recovering the secondary metabolite from the culture broth.

In the production methods of the present invention, the cells arecultivated in a nutrient medium suitable for production of thepolypeptide or metabolite using methods known in the art. For example,the cell may be cultivated by shake flask cultivation, small-scale orlarge-scale fermentation (including continuous, batch, fed-batch, orsolid state fermentations) in laboratory or industrial fermentorsperformed in a suitable medium and under conditions allowing the codingsequence to be expressed and/or the polypeptide to be isolated. Thecultivation takes place in a suitable nutrient medium comprising carbonand nitrogen sources and inorganic salts, using procedures known in theart. Suitable media are available from commercial suppliers or may beprepared according to published compositions (e.g., in catalogues of theAmerican Type Culture Collection). If the polypeptide or metabolite issecreted into the nutrient medium, the polypeptide or metabolite can berecovered directly from the medium. If the polypeptide or metabolite isnot secreted, it can be recovered from cell lysates.

The polypeptides may be detected using methods known in the art that arespecific for the polypeptides. These detection methods may include useof specific antibodies, formation of an enzyme product, or disappearanceof an enzyme substrate.

The resulting polypeptide or metabolite may be recovered by methodsknown in the art. For example, the polypeptide or metabolite may berecovered from the nutrient medium by conventional procedures including,but not limited to, centrifugation, filtration, extraction,spray-drying, evaporation, or precipitation.

Polypeptides may be purified by a variety of procedures known in the artincluding, but not limited to, chromatography (e.g., ion exchange,affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, or extraction (see, e.g., Protein Purification, J.-C. Jansonand Lars Ryden, editors, VCH Publishers, New York, 1989).

The present invention also relates to DNA constructs for altering theexpression of a coding sequence encoding a polypeptide, which isendogenous to a fungal host cell. The constructs may contain the minimalnumber of components necessary for altering expression of the endogenousgene.

In one embodiment, the nucleic acid constructs preferably contain (a) atargeting sequence, (b) a promoter DNA sequence of the presentinvention, (c) an exon, and (d) a splice-donor site. Upon introductionof the nucleic acid construct into a cell, the construct integrates byhomologous recombination into the cellular genome at the endogenous genesite. The targeting sequence directs the integration of elements (a)-(d)into the endogenous gene such that elements (b)-(d) are in operativeassociation with the endogenous gene.

In another embodiment, the nucleic acid constructs contain (a) atargeting sequence, (b) a promoter DNA sequence of the presentinvention, (c) an exon, (d) a splice-donor site, (e) an intron, and (f)a splice-acceptor site, wherein the targeting sequence directs theintegration of elements (a)-(f) such that elements (b)-(f) are inoperative association with the endogenous gene. However, the constructsmay contain additional components such as a selectable marker. Theselectable markers that can be used were earlier described.

In both embodiments, the introduction of these components results inproduction of a new transcription unit in which expression of theendogenous gene is altered. In essence, the new transcription unit is afusion product of the sequences introduced by the targeting constructsand the endogenous gene. In one embodiment in which the endogenous geneis altered, the gene is activated. In this embodiment, homologousrecombination is used to replace, disrupt, or disable the regulatoryregion normally associated with the endogenous gene of a parent cellthrough the insertion of a regulatory sequence, which causes the gene tobe expressed at higher levels than evident in the corresponding parentcell.

The targeting sequence can be within the endogenous gene, immediatelyadjacent to the gene, within an upstream gene, or upstream of and at adistance from the endogenous gene. One or more targeting sequences canbe used. For example, a circular plasmid or DNA fragment preferablyemploys a single targeting sequence, while a linear plasmid or DNAfragment preferably employs two targeting sequences.

The constructs further contain one or more exons of the endogenous gene.An exon is defined as a DNA sequence, which is copied into RNA and ispresent in a mature mRNA molecule such that the exon sequence isin-frame with the coding region of the endogenous gene. The exons can,optionally, contain DNA, which encodes one or more amino acids and/orpartially encodes an amino acid. Alternatively, the exon contains DNAwhich corresponds to a 5′ non-encoding region. Where the exogenous exonor exons encode one or more amino acids and/or a portion of an aminoacid, the nucleic acid construct is designed such that, upontranscription and splicing, the reading frame is in-frame with thecoding region of the endogenous gene so that the appropriate readingframe of the portion of the mRNA derived from the second exon isunchanged. The splice-donor site of the constructs directs the splicingof one exon to another exon. Typically, the first exon lies 5′ of thesecond exon, and the splice-donor site overlapping and flanking thefirst exon on its 3′ side recognizes a splice-acceptor site flanking thesecond exon on the 5′ side of the second exon. A splice-acceptor site,like a splice-donor site, is a sequence, which directs the splicing ofone exon to another exon. Acting in conjunction with a splice-donorsite, the splicing apparatus uses a splice-acceptor site to effect theremoval of an intron.

A preferred strategy for altering the expression of a given DNA sequencecomprises the deletion of the given DNA sequence and/or replacement ofthe endogenous promoter sequence of the given DNA sequence by a modifiedpromoter DNA sequence, such as a promoter of the invention. The deletionand the replacement are preferably performed by the gene replacementtechnique described in EP 0 357 127. The specific deletion of a geneand/or promoter sequence is preferably performed using the amdS gene asselection marker gene as described in EP 635 574. By means ofcounterselection on fluoroacetamide media as described in EP 635 574,the resulting strain is selection marker free and can be used forfurther gene modifications.

Alternatively or in combination with other mentioned techniques, atechnique based on in vivo recombination of cosmids in E. coli can beused, as described in: A rapid method for efficient gene replacement inthe filamentous fungus A. nidulans (2000) Chaveroche, M-K., Ghico, J-M.and d'Enfert C; Nucleic acids Research, vol 28, no 22. This technique isapplicable to other filamentous fungi like for example A. niger. Theinvention described and claimed herein is not to be limited in scope bythe specific embodiments herein disclosed, since these embodiments areintended as illustrations of several aspects of the invention. Anyequivalent embodiments are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

All patents and publications, including all sequences and methodsdisclosed within such patents and publications, referred to herein areexpressly incorporated by reference. These patents and publicationsinclude: EP 357 127, EP 673 429, EP 635 574, WO 97/06261, WO 98/46772,WO 94/04673.

EXAMPLES Experimental Information Strains

WT 1: This A. niger strain is used as a wild-type strain. This strain isdeposited at the CBS Institute under the deposit number CBS 513.88.

WT 2: This A. niger strain is a WT 1 strain comprising a deletion of thegene encoding glucoamylase (glaA). WT 2 was constructed by using the“MARKER-GENE FREE” approach as described in EP 0 635 574. In this patentit is extensively described how to delete glaA specific DNA sequences inthe genome of CBS 513.88. The procedure resulted in a MARKER-GENE FREE?glaA recombinant A. niger CBS513.88 strain, possessing finally noforeign DNA sequences at all.

Glucoamylase Activity Assay

The glucoamylase activity was determined using p-Nitrophenyla-D-glucopyranoside (Sigma) as described in WO 98/46772.

Example 1 Construction of a DNA Construct Comprising a Promoter of theInvention in Operative Association with a Coding Sequence

This example describes the construction of an expression construct undercontrol of a promoter of the invention. The coding sequence or reporterconstruct used here is the glaA gene encoding the A. niger glucoamylaseenzyme. Glucoamylase is used as the reporter enzyme to be able tomeasure the activity of the promoter of the invention.

1.1 Description of an Integrative Glucoamylase Expression Vector(pGBTOPGLA)

The glucoamylase promoter and the glucoamylase encoding gene glaA fromA. niger were cloned into the expression vector pGBTOP-8, which isdescribed in WO99/32617. The cloning was performed according knownprinciples and to routine cloning techniques and yielded plasmidpGBTOPGLA (see FIG. 1). In essence, this expression vector comprises theglucoamylase promoter, coding sequence and terminator region, flanked bythe 3′ and 3″ glaA targeting sites in an E. coli vector.

1.2 Construction of an Integrative Glucoamylase Expression Vector with aMultiple Cloning Site MCS (pGBTOPGLA-2)

Using the oligonucleotides

5′-ATgCggCCgCCTCgAgTTAATTAAggCCAggCCggCCggCgCgCCTCAgCAATgTCgTTC CgA-3′identified as SEQ ID NO 6 and

5′-AGCCATTGACTTCTTCCCAG-3′

identified as SEQ ID NO 7 and 1 ng of vector pGBTOPGLA as a template, aPCR fragment was generated containing part of the glaA coding sequence.This fragment was digested with XhoI and BglII and introduced in XhoIand Bg/II digested vector pGBTOPGLA, resulting in vector pGBTOPGLA-2(see FIG. 2). The sequence of the introduced PCR fragment comprising aMCS and part of the glaA coding sequence was confirmed by sequenceanalysis.

1.3 Construction of an Integrative Expression Vector with the Promoterof the Invention in Operative Association with the Glucoamylase CodingSequence (pGBTOPGLA-3)

Genomic DNA of strain CBS513.88 was sequenced and analysed. Using theoligonucleotide combinations as identified below (Oligo SEQ ID NO's) inthe Table and genomic DNA of strain CBS513.88 as template, appropriaterestriction sites were attached to the promoter of the invention by PCRamplification. The sequences as identified in SEQ ID NO 1, SEQ ID NO:2,SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5 comprise the sequences of theresulting fragments of about 2 kb, as indicated in the Table below. Allfive resulting fragments were digested with AscI and XhoI and introducedin AscI and XhoI digested vector pGBTOPGLA-2, resulting in vectorpGBTOPGLA-4, pGBTOPGLA-5, pGBTOPGLA-7, GBTOPGLA-9 or pGBTOPGLA-10, asindicated in the Table (Vector name). In FIG. 3, a picture can be foundwhich is illustrative for the layout of all five vectors. The sequenceof the various introduced PCR fragments comprising the promoters of theinvention was confirmed by sequence analysis.

Oligo Oligo SEQ ID NO: SEQ ID SEQ ID of promoter cloned NO: NO: Vectorname in vector 8 9 pGBTOPGLA-7 1 10 11 pGBTOPGLA-9 2 12 13 pGBTOPGLA-4 314 15 pGBTOPGLA-10 4 16 17 pGBTOPGLA-5 5

Example 2 Fungal Host Cell Transformed with the DNA Construct

In order to introduce the pGBTOPGLA, pGBTOPGLA-4, pGBTOPGLA-5,pGBTOPGLA-7, pGBTOPGLA-9 or pGBTOPGLA-10 vectors in WT 2, atransformation and subsequent transformant selection was carried out asdescribed in WO98/46772 and WO99/32617. In principle, linear DNA of allvectors was isolated after digestion with NotI and co-transformed withan amdS selectable marker-gene containing vector, which is designatedpGBAAS-1 (constructed as described in EP 635574). Both vectors comprisetwo DNA domains homologous to the glaA locus of A. niger host strain todirect targeting to the truncated glaA locus in WT 2. Transformants wereselected on acetamide media and colony purified according standardprocedures. Spores were plated on fluoro-acetamide media to selectstrains, which lost the amdS marker. Growing colonies were diagnosed forintegration at the glaA locus and copy number. Transformants ofpGBTOPGLA, pGBTOPGLA-4, pGBTOPGLA-5, pGBTOPGLA-7, pGBTOPGLA-9 orpGBTOPGLA-10 with similar estimated copy numbers were selected.Preferably, few transformants with a single copy (1A, 1B, 1) andpossibly one with multiple copies (2A) were selected.

Additionally, the selectable marker gene and the gene of interestcontrolled by a promoter of the invention would have been on oneconstruct. An example of this is shown in FIG. 4.

Example 3 Production of the Glucoamylase Polypeptide Encoded by the glaACoding Sequence Under Control of a Promoter of the Invention in theFungal Host Cell

A number of selected transformants of WT 2, as described above, and bothstrains WT 1 and WT 2 were used to perform shake flask experiments in100 ml of the medium as described in EP 635 574 at 34° C. and 170 rpm inan incubator shaker using a 500 ml baffled shake flask. After 4 and 5days of fermentation, samples were taken to determine the glucoamylaseactivity, as described above. The glucoamylase activities werenormalized to the activity of WT 1 on day 4. The normalized activitiesof WT 1, WT 2 and a number of selected transformants for of pGBTOPGLA,pGBTOPGLA-5, pGBTOPGLA-7, and pGBTOPGLA-10 are indicated in FIG. 5

As can be concluded from the measured activities of the glucoamylasereporter, the invention provides a strong promoter for high expressionof a gene of interest in a fungal cell. As such, the promoters of theinvention provide alternative and additional promoters for highexpression of a gene of interest in a fungal cell.

Example 4 Construction of a Promoter Replacement Construct PGBDEL-PGLAAComprising a Promoter of the Invention

To alter the expression level of a given gene in a host cell, a promoterof the invention can replace the endogenous promoter of said given gene.In this example, a promoter of the invention replaces the promoter ofthe glucoamylase encoding glaA gene in a fungal host cell. Examples 4, 5and 6 describe a number of different steps in this process.

A replacement vector for the glucoamylase promoter was designedaccording to known principles and constructed according to routinecloning procedures (see FIG. 6). In essence, the glaA promoterreplacement vector pGBDEL-PGLAA comprises approximately 1000 bp flankingregions of the glaA promoter sequence to be replaced by a promoter ofthe invention, which is comprised by SEQ ID NO: 4, trough homologousrecombination at the predestined genomic locus. The flanking regionsused here (see FIG. 6) are a 5′ upstream region of the glaA promoter andpart of the glaA coding sequence. In addition, the replacement vectorcontains the A. nidulans bi-directional amdS selection marker,in-between direct repeats. The direct repeats used in this example arepart of the glaA coding sequence. The general design of these deletionvectors were previously described in EP635574 and WO 98/46772.

Example 5 Replacement of the glaA Promoter by a Promoter of theInvention in the Fungal Host Cell

Linear DNA of NotI-digested deletion vector pGBDEL-PGLAA was isolatedand used to transform WT 1 (CBS513.88). This linear DNA can integrateinto the genome at the glaA locus, thus substituting the glaA promoterregion with the construct containing amdS and a promoter of theinvention (see FIG. 7). Transformants were selected on acetamide mediaand colony purified according to standard procedures. Growing colonieswere diagnosed by PCR for integration at the glaA locus. Deletion of theglaA promoter was detectable by amplification of a band, with a sizespecific for the promoter of the invention and loss of a band specificfor the glaA promoter. Spores were plated on fluoro-acetamide media toselect strains, which lost the amdS marker. Candidate strains weretesting using Southern analysis for proper deletion of the glucoamylasepromoter and replacement by a promoter of the invention, as comprised bySEQ ID NO: 4. Strains dPGLAA were selected as representative strainswith the glaA promoter replaced by the promoter of the invention andhaving a restored functional glaA coding sequence (see FIG. 7).

Example 6 Production of the Glucoamylase Polypeptide Encoded by the glaACoding Sequence Under Control of a Replaced Promoter of the Invention,in the Fungal Host Cell

The selected dPGLAA strains (proper pGBDEL-PGLAA transformants of WT 1,isolated in example 5) and strain WT 1 were used to perform shake flaskexperiments in 100 ml of the medium as described in EP 635 574 B1 at 34°C. and 170 rpm in an incubator shaker using a 500 ml baffled shakeflask. Further conditions and activity measurements were as described inExample 3. The glucoamylase activity in the selected pGBDEL-PGLAAtransformants of WT1 was increased compared to the one measured for WT 1at both days of fermentation (data not shown).

Example 7 Addition of an Additional glaA Gene Under Control of aPromoter of the Invention in the Fungal Host Cell

To alter the expression level of a given gene in a host cell, multipleadditional copies of said gene operatively linked to a promoter of theinvention can be added to the endogenously given gene. In this example,a promoter of the invention, as comprised by SEQ ID NO: 4 andoperatively linked with the glaA coding sequence is introduced next tothe endogenously present glucoamylase encoding glaA gene in a fungalhost cell. Example 7 and 8 describe a number of different steps in thisprocess.

A circular construct as depicted in FIG. 8 was isolated and used totransform WT 1 (CBS513.88). This linear DNA can integrate into thegenome at the glaA coding sequence, thus adding a second glaA gene undercontrol of a promoter of the invention next to the selectable markeramdS (see FIG. 8). Transformants were selected on acetamide media andcolony purified according to standard procedures. Growing colonies werediagnosed by PCR for integration at the glaA locus. Integration at theglaA locus was detected by PCR and Southern analysis. Strains P2GLAAwere selected as representative strains with at least a second glaA geneunder control of a promoter of the invention integrated at the glaAlocus.

Example 8 Production of the Glucoamylase Polypeptide Encoded by the glaACoding Sequences Under Control of a Promoter of the Invention and theEndogenous glaA Promoter in the Fungal Host Cell

The selected P2GLAA strains, isolated in example 7, and strain WT 1 wereused to perform shake flask experiments in 100 ml of the medium asdescribed in Example 3. After 4 and 6 days of fermentation, samples weretaken to determine the glucoamylase activity. The glucoamylase activityin the selected P2GLAA transformants of WT1 was increased compared tothe one measured for WT 1 after either four or five days offermentation. The increased activities of the glucoamylase reporterindicate that the promoters of the invention provide a means to furtherincrease the expression of a gene of interest which is already expressedunder a strong promoter in a fungal cell.

1. An isolated DNA sequence selected from the group consisting of: (a) a DNA sequence comprising SEQ ID NO:1, (b) a DNA sequence capable of hybridizing to the complement of the full length sequence of SEQ ID NO:1 under high stringency conditions of 42° C., 5×SSPE, 0.3% SDS, 200 μg/ml sheared and denatured salmon sperm DNA, and 50% formamide followed by washing three times each for 15 minutes using 2×SSC, 0.2% SDS at 60° C., and (c) a DNA sequence that is at least 90% identical to SEQ ID NO:1 along the full length of SEQ ID NO:1; said isolated DNA sequence having promoter activity.
 2. A recombinant DNA construct comprising a DNA sequence according to claim 1 and a coding sequence in operative association with said DNA sequence such that the coding sequence can be expressed under control of the DNA sequence.
 3. A host cell comprising the DNA construct according to claim
 2. 4. The host cell according to claim 3, wherein the host cell is an Aspergillus, Penicillium or Trichoderma species.
 5. The host cell according to claim 4, wherein the host cell is an Aspergillus niger or Aspergillus oryzae species.
 6. A method for expression of a gene comprising: (a) providing the DNA construct according to claim 2 wherein the coding sequence comprises a gene, (b) transforming a suitable host cell with said DNA construct, and (c) expressing the gene under control of said DNA sequence in the transformed host cell.
 7. A method for production of a polypeptide comprising: (a) providing the DNA construct according to claim 2 comprising a coding sequence encoding the polypeptide, (b) transforming a suitable host cell with said DNA construct, (c) culturing the transformed host cell under conditions conducive to expression of the polypeptide, and (d) recovering the polypeptide from culture broth.
 8. A method for production of a secondary metabolite comprising: (a) providing the DNA construct according to claim 2 comprising a coding sequence encoding an enzyme involved in the production of a secondary metabolite, (b) transforming a suitable host cell with said DNA construct, (c) culturing the transformed host cell under conditions conducive to production of the secondary metabolite, and (d) recovering the secondary metabolite from culture broth.
 9. The isolated DNA sequence of claim 1 wherein said sequence is the DNA sequence comprising SEQ ID NO:1.
 10. A recombinant expression vector comprising (i) a DNA sequence according to claim 1, (ii) a coding sequence in operative association with said DNA sequence such that the coding sequence can be expressed under control of the DNA sequence as a promoter and (iii) transcriptional and translational stop signals.
 11. A host cell comprising the expression vector according to claim
 10. 12. A method for expression of a gene comprising: (a) providing the expression vector according to claim 10 wherein the coding sequence comprises a gene, (b) transforming a suitable host cell with said expression vector, and (c) expressing the gene under control of said promoter in the transformed host cell.
 13. A method for production of a polypeptide comprising: (a) providing the expression vector according to claim 10 comprising a coding sequence encoding the polypeptide, (b) transforming a suitable host cell with said expression vector, (c) culturing the transformed host cell under conditions conducive to expression of the polypeptide, and (d) recovering the polypeptide from culture broth.
 14. A method for production of a secondary metabolite comprising: (a) providing the expression vector according to claim 10 comprising a coding sequence encoding an enzyme involved in the production of a secondary metabolite, (b) transforming a suitable host cell with said expression vector, (c) culturing the transformed host cell under conditions conducive to production of the secondary metabolite, and (d) recovering the secondary metabolite from culture broth.
 15. A recombinant DNA construct comprising a DNA sequence according to claim 9 and a coding sequence in operative association with said DNA sequence such that the coding sequence can be expressed under control of the DNA sequence.
 16. A host cell comprising the DNA construct according to claim
 15. 17. The host cell according to claim 16, wherein the host cell is an Aspergillus, Penicillium or Trichoderma species.
 18. The host cell according to claim 17, wherein the host cell is an Aspergillus niger or Aspergillus oryzae species.
 19. A method for expression of a gene comprising: (a) providing the DNA construct according to claim 15 wherein the coding sequence comprises a gene, (b) transforming a suitable host cell with said DNA construct, and (c) expressing the gene under control of said DNA sequence in the transformed host cell.
 20. A method for production of a polypeptide comprising: (a) providing the DNA construct according to claim 15 comprising a coding sequence encoding the polypeptide, (b) transforming a suitable host cell with said DNA construct, (c) culturing the transformed host cell under conditions conducive to expression of the polypeptide, and (d) recovering the polypeptide from culture broth.
 21. A method for production of a secondary metabolite comprising: (a) providing the DNA construct according to claim 15 comprising a coding sequence encoding an enzyme involved in the production of a secondary metabolite, (b) transforming a suitable host cell with said DNA construct, (c) culturing the transformed host cell under conditions conducive to production of the secondary metabolite, and (d) recovering the secondary metabolite from culture broth.
 22. A recombinant expression vector comprising (i) a DNA sequence according to claim 9, (ii) a coding sequence in operative association with said DNA sequence such that the coding sequence can be expressed under control of the DNA sequence as a promoter and (iii) transcriptional and translational stop signals.
 23. A host cell comprising the expression vector according to claim
 22. 24. A method for expression of a gene comprising: (a) providing the expression vector according to claim 22 wherein the coding sequence comprises a gene, (b) transforming a suitable host cell with said expression vector, and (c) expressing the gene under control of said promoter in the transformed host cell.
 25. A method for production of a polypeptide comprising: (a) providing the expression vector according to claim 22 comprising a coding sequence encoding the polypeptide, (b) transforming a suitable host cell with said expression vector, (c) culturing the transformed host cell under conditions conducive to expression of the polypeptide, and (d) recovering the polypeptide from culture broth.
 26. A method for production of a secondary metabolite comprising: (a) providing the expression vector according to claim 22 comprising a coding sequence encoding an enzyme involved in the production of a secondary metabolite, (b) transforming a suitable host cell with said expression vector, (c) culturing the transformed host cell under conditions conducive to production of the secondary metabolite, and (d) recovering the secondary metabolite from culture broth. 